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Label-Free and Simultaneous Mechanical and Electrical Characterization of Single Plant Cells Using Microfluidic Impedance Flow Cytometry
Abstract
Despite that single-cell-type-level analyses have been extensively conducted on animal models to gain new insight into complex biological processes, the unique biological and physiological properties of plant cells have not been widely studied at single-cell resolution. In this work, an electrical impedance flow cytometry was fabricated based on microfluidics with constriction microchannel to simultaneously characterize the mechanical and electrical properties of single plant cells. Protoplasts from two model plant species, the herbaceous Arabidopsis thaliana and the woody Populus trichocarpa, could be readily discriminated by their respective mechanical traits, but not by electrical impedance. On the contrary, overexpression of a red fluorescent protein (RFP) on plasma membrane resulted in changes in cell electrical impedance instead of cell deformability. During primary cell wall (PCW) regeneration, this extracellular layer outside of protoplasts introduced dramatic variations in both mechanical and electrical properties of single plant cells. Furthermore, the effects of auxin, an essential phytohormone regulating PCW reformation, were validated on this platform. Taken together, our results revealed a novel application of microfluidic impedance flow cytometry in the field of plant science to simultaneously characterize dual biophysical properties at single cell resolution, which could be further developed as a powerful and reliable tool for plant cell phenotyping and cell fate specification.
... Upon inhalation, these particles can accumulate in the trachea or lungs, potentially affecting respiratory health. PM exposure is associated with morbidity and mortality from respiratory and cardiovascular diseases (Guo et al., 2022;Han et al., 2020;Losacco and Perillo, 2018;Yu et al., 2022). Although most of the inhaled airborne particles are cleared by airway mucociliary systems, particles with aerodynamic diameter below 5 μm may penetrate and retain in the respiratory system (Grzywa-Celinska et al., 2020;Guo et al., 2022). ...
... Microfluidic chip setup was described as previously (Han et al., 2020). In brief, two pairs of electrodes were symmetrically placed at the entrance and exit of the constriction channel, which is 8 μm in width, 20 μm in height, and 600 μm in length. ...
... The electrical and mechanical properties can be extracted from real-time electrical signals. The amplitude of the pulse signals is defined as the impedance of the cells, and the time interval between the negative and positive pulses is defined as the passage time, which is related to the mechanical properties because cells with poor deformability need to spend more time to deform themselves to squeeze into a constriction channel that is smaller than their diameter (Han et al., 2020). These changes reflected different information with changes in the frequency of the excitation signal. ...
... Constriction channel allows for more accurate detection of specific membrane capacitance and cytoplasm conductivity, thus increasing the classification success rate of different cell populations [58]. Hence, it has been used for screening various types of single cells, such as blood cells [59], tumor cells [43,58] and plant cells [60]. Due to the cross-sectional area of the constriction channel must be smaller than the size of the interested cells, this design has higher risk of channel blockage and lower throughput. ...
... The passage time for cells to pass through the constriction channel is related to cellular mechanical properties. In order to obtain the passage time, Han et al. introduced a pairs of coplanar electrodes at the inlet and outlet of the constriction channel [60]. The time point when a cell passes through each sensing unit is recorded, and then the passage time can be calculated according to the time interval for a cell from a sensing unit to another. ...
... Recent IFC devices applied in single-cell analysis are summarized in Table 1. These applications, discussed in this subsection, are simply classified according to cell species, including blood cells [110][111][112][113][114], tumor cells [43,52,[115][116][117][118][119][120][121][122], stem cells [123][124][125][126][127], plant cells [60,[128][129][130][131][132] and microbes [53,62,[133][134][135][136][137][138][139][140][141]. In terms of blood cells, researchers focused on the identification and counting of normal or diseased blood cells. ...
Cellular heterogeneity is of significance in cell-based assays for life science, biomedicine and clinical diagnostics. Electrical impedance sensing technology has become a powerful tool, allowing for rapid, non-invasive, and label-free acquisition of electrical parameters of single cells. These electrical parameters, i.e., equivalent cell resistance, membrane capacitance and cytoplasm conductivity, are closely related to cellular biophysical properties and dynamic activities, such as size, morphology, membrane intactness, growth state, and proliferation. This review summarizes basic principles, analytical models and design concepts of single-cell impedance sensing devices, including impedance flow cytometry (IFC) to detect flow-through single cells and electrical impedance spectroscopy (EIS) to monitor immobilized single cells. Then, recent advances of both electrical impedance sensing systems applied in cell recognition, cell counting, viability detection, phenotypic assay, cell screening, and other cell detection are presented. Finally, prospects of impedance sensing technology in single-cell analysis are discussed.
... More recently, AC methods have been used to determine both the size and properties of the cell membrane, such as cytoplasm and subcellular components. [3][4][5][6][7][8] In general, impedance cytometry of a cell is carried out in a microfluidic channel based on the impedance signal when the cell-suspended in a conductive solution-passes through microelectrodes fabricated on the inner walls of the channel, and accurate determination of particle size has been explored with different microelectrode configurations. [9][10][11][12] The literature contains two different microelectrode configurations, i.e., (i) coplanar, where the electrodes are etched on one wall of the channel with rectangular cross section, and (ii) parallel facing, where the electrodes are located on opposite walls of the channel. ...
... 8(c) and 8(d), and the comparative results are provided in Table II for different particle sizes. From Table II, for the separate runs it is clear that there was a reduction in the standard deviation by a factor of two irrespective of particle diameter, which indicates the validity of the empirical relationship in Eq. (5). The experimental data from the mixed runs show again the applicability of Eq. (5), where both the standard deviation and coefficient of variation (CV) were found to ...
HIGHLIGHTS
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Measurement of particle size through microfluidic-based impedance cytometry using parallel facing electrodes.
•
Development of an analytical formula for a rectangular channel via 3D COMSOL simulation.
•
Implementation of the analytical formula for accurate determination of particle size irrespective of value.
ABSTRACT
In microfluidic impedance cytometry, the change in impedance is recorded as an individual cell passes through a channel between electrodes deposited on its walls, and the particle size is inferred from the amplitude of the impedance signal using calibration. However, because the current density is nonuniform between electrodes of finite width, there could be an error in the particle size measurement because of uncertainty about the location of the particle in the channel cross section. Here, a correlation is developed relating the particle size to the signal amplitude and the velocity of the particle through the channel. The latter is inferred from the time interval between the two extrema in the impedance curve as the particle passes through a channel with cross-sectional dimensions of 50 μm (width) × 30 μm (height) with two pairs of parallel facing electrodes. The change in impedance is predicted using 3D COMSOL finite-element simulations, and a theoretical correlation that is independent of particle size is formulated to correct the particle diameter for variations in the cross-sectional location. With this correlation, the standard deviation in the experimental data is reduced by a factor of two to close to the standard deviation reported in the manufacturer specifications.
... Using passive force to aspirate the individual cells sequentially into a constriction with a crosssection area smaller than the cell size, the cell can be confined within the constriction, allowing the impedance measurement of single cells. By integrating a straight constriction microchannel between two pairs of electrodes to form an impedance sensing platform, Han et al. characterized the electrical and mechanical properties of individual plant cells simultaneously by analyzing the impedance response and the transit time difference of the deformed cell passing through the constriction channel [82]. The flexible design of constriction structures and reliable circuit models enable the extraction of biophysical properties such as membrane capacitance, cytoplasm conductivity, and viscosity [110]. ...
Cellular heterogeneity plays a significant role in understanding biological processes, such as cell cycle and disease progression. Microfluidics has emerged as a versatile tool for manipulating single cells and analyzing their heterogeneity with the merits of precise fluid control, small sample consumption, easy integration, and high throughput. Specifically, integrating microfluidics with electrical techniques provides a rapid, label-free, and non-invasive way to investigate cellular heterogeneity at the single-cell level. Here, we review the recent development of microfluidic-based electrical strategies for single-cell manipulation and analysis, including dielectrophoresis- and electroporation-based single-cell manipulation, impedance- and AC electrokinetic-based methods, and electrochemical-based single-cell detection methods. Finally, the challenges and future perspectives of the microfluidic-based electrical techniques for single-cell analysis are proposed.
... Furthermore, by controlling the flow bed of the reagent in the microreactor, the sorting of cells can be realized, and the labeling treatment of cells is omitted. Hand et al. reported a microfluidic resistance flow cytometer for studying the special biological and physiological properties of individual plant cells ( Figure 5A), which can be used to characterize the mechanical and electrical characteristics in a single plant cell at the same time, and also to verify the role of auxin [107]. ...
In recent years, with the outbreak of the global energy crisis, renewable solar energy has become a focal point of research. However, the utilization efficiency of natural photosynthesis (NPS) is only about 1%. Inspired by NPS, artificial photosynthesis (APS) was developed and utilized in applications such as the regeneration of coenzymes. APS for coenzyme regeneration can overcome the problem of high energy consumption in comparison to electrocatalytic methods. Microreactors represent a promising technology. Compared with the conventional system, it has the advantages of a large specific surface area, the fast diffusion of small molecules, and high efficiency. Introducing microreactors can lead to more efficient, economical, and environmentally friendly coenzyme regeneration in artificial photosynthesis. This review begins with a brief introduction of APS and microreactors, and then summarizes research on traditional electrocatalytic coenzyme regeneration, as well as photocatalytic and photo-electrocatalysis coenzyme regeneration by APS, all based on microreactors, and compares them with the corresponding conventional system. Finally, it looks forward to the promising prospects of this technology.
... The observed peak values were not uniform, which can be attributed to the intrinsic heterogeneity of the cells. Different cell morphologies or sizes with varying contact areas with the microelectrode could lead to different impedance values [61]. Overall, this microfluidic device enables reliable single-cell analysis based on the electrical properties of cells and has great potential for real-time cell monitoring. ...
In the context of wound healing and tissue regeneration, precise control of cell migration direction is deemed crucial. To address this challenge, polydimethylsiloxane (PDMS) platforms with patterned 10 nm thick TiOx in arrowhead shape were designed and fabricated. Remarkably, without tall sidewall constraints, MC3T3-E1 cells seeded on these platforms were constrained to migrate along the tips of the arrowheads, as the cells were guided by the asymmetrical arrowhead tips which provided large contact areas. To the best of our knowledge, this is the first study demonstrating the use of thin TiOx arrowhead pattern in combination with a cell-repellent PDMS surface to provide guided cell migration unidirectionally without tall sidewall constraints. Additionally, high-resolution fluorescence imaging revealed that the asymmetrical distribution of focal adhesions, triggered by the patterned TiOx arrowheads with arm lengths of 10, 20, and 35 μm, promoted cell adhesion and protrusion along the arrowhead tip direction, resulting in unidirectional cell migration. These findings have important implications for the design of biointerfaces with ultrathin patterns to precisely control cell migration. Furthermore, microelectrodes were integrated with the patterned TiOx arrowheads to enable dynamic monitoring of cell migration using impedance measurement. This microfluidic device integrated with thin layer of guiding pattern and microelectrodes allows simultaneous control of directional cell migration and characterization of the cell movement of individual MC3T3-E1 cells, offering great potential for the development of biosensors for single-cell monitoring.
... In this design, electrodes are arranged before and after the contraction channel, allowing for the indirect extraction of time-related parameters by detecting the time point of impedance amplitude mutation. It enables label-free, real-time, and high-throughput measurements while streamlining data extraction and processing [58,146]. Zhou et al. [33] developed a microfluidic platform that incorporates four pairs of coplanar electrodes located at the bottom of the channel, utilizing differential impedance measurement. ...
Single‐cell biophysical properties play a crucial role in regulating cellular physiological states and functions, demonstrating significant potential in the fields of life sciences and clinical diagnostics. Therefore, over the last few decades, researchers have developed various detection tools to explore the relationship between the biophysical changes of biological cells and human diseases. With the rapid advancement of modern microfabrication technology, microfluidic devices have quickly emerged as a promising platform for single‐cell analysis offering advantages including high‐throughput, exceptional precision, and ease of manipulation. Consequently, this paper provides an overview of the recent advances in microfluidic analysis and detection systems for single‐cell biophysical properties and their applications in the field of cancer. The working principles and latest research progress of single‐cell biophysical property detection are first analyzed, highlighting the significance of electrical and mechanical properties. The development of data acquisition and processing methods for real‐time, high‐throughput, and practical applications are then discussed. Furthermore, the differences in biophysical properties between tumor and normal cells are outlined, illustrating the potential for utilizing single‐cell biophysical properties for tumor cell identification, classification, and drug response assessment. Lastly, we summarize the limitations of existing microfluidic analysis and detection systems in single‐cell biophysical properties, while also pointing out the prospects and future directions of their applications in cancer diagnosis and treatment.
... Other researchers have introduced a microfluidic with a constriction channel, which is a simple and high throughput technique to determine the electrical characteristics of a single cell (e.g. specific membrane capacitance) [16][17][18][19]. However, the microfluidic device with a constriction channel approach is difficult to optimize for sizeheterogeneous samples. ...
Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially available tungsten needle coated with parylene. When a single cell flows through the parallel-facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells (RBCs) with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m⁻², with the regression coefficients, ρ at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.
... The key advantage of this method is optics-free, but the major disadvantage is that it could be a challenge to detect subtle morphological changes due to the impedance variation with respect to size and position. Another method applies a constriction structure to measure the cell transit time as the cell deformability characterization, and meanwhile extract the impedance information when the cell passes through electrodes (Han et al., 2020). With the concurrent mechanical and electrical phenotyping method, the subtypes of plant cells could be classified. ...
Microfluidics provides a powerful platform for biological analysis by harnessing the ability to precisely manipulate fluids and microparticles with integrated microsensors. Here, we introduce an imaging and impedance cell analyzer (IM2Cell), which implements single cell level impedance analysis and hydrodynamic mechanical phenotyping simultaneously. For the first time, IM2Cell demonstrates the capability of multi-stress level mechanical phenotyping. Specifically, IM2Cell is capable of characterizing cell diameter, three deformability responses, and four electrical properties. It presents high-dimensional information to give insight into subcellular components such as cell membrane, cytoplasm, cytoskeleton, and nucleus. In this work, we first validate imaging and impedance-based cell analyses separately. Then, the two techniques are combined to obtain both imaging and impedance data analyzed by machine learning method, exhibiting an improved prediction accuracy from 83.1% to 95.4% between fixed and living MDA-MB-231 breast cancer cells. Next, IM2Cell demonstrates 91.2% classification accuracy in a mixture of unlabeled MCF-10A, MCF-7, and MDA-MB-231 cell lines. Finally, an application demonstrates the potential of IM2Cell for the deformability studies of peripheral blood mononuclear cells (PBMCs) subpopulations without cumbersome isolation or labeling steps.
... In order to further improve the performance of the system, future work could try some other impedance metrics, in addition to impedance magnitudes and phases; for example, it has been [14] reported that the impedance opacity is sensitive to the changes in drug-treated bacterial cells. Therefore, we think that the recognition accuracy of the system could be improved by using more sensitive dielectric properties, such as tilt index [18,32], pulse duration [35,53], and impedance opacity [46,54]. Additionally, the maximum throughput of the system is around 1264 particles/s, due to the time required for single particle recognition (0.791 ± 0.078 ms). ...
Impedance cytometry has enabled label-free and fast antibiotic susceptibility testing of bacterial single cells. Here, a machine learning-based impedance system is provided to score the phenotypic response of bacterial single cells to antibiotic treatment, with a high throughput of more than one thousand cells per min. In contrast to other impedance systems, an online training method on reference particles is provided, as the parallel impedance cytometry can distinguish reference particles from target particles, and label reference and target particles as the training and test set, respectively, in real time. Experiments with polystyrene beads of two different sizes (3 and 4.5 μm) confirm the functionality and stability of the system. Additionally, antibiotic-treated Escherichia coli cells are measured every two hours during the six-hour drug treatment. All results successfully show the capability of real-time characterizing the change in dielectric properties of individual cells, recognizing single susceptible cells, as well as analyzing the proportion of susceptible cells within heterogeneous populations in real time. As the intelligent impedance system can perform all impedance-based characterization and recognition of particles in real time, it can free operators from the post-processing and data interpretation.
... Moreover, multifrequency impedance measurements in the 500 kHz-20 MHz range could be simultaneously performed, providing cell characterization in terms of biophysical properties such as cell membrane capacitance and cytoplasm conductivity [50], [51]. Indeed, a multimodal analysis based on different kinds of cellular biomarkers, such as mechanical and electrical properties, may provide better discrimination of cellular phenotypes compared to single-marker analysis [23], [24], [52], [53], [54], [55]. ...
Objective:
Deformability is an essential feature of red blood cells (RBCs), enabling them to undergo significant shape change in response to external forces. Impaired erythrocyte deformability is associated with several pathologic conditions, and quantitative measurement of RBC deformability is critical to understanding and diagnosing RBC related diseases. Whereas traditional approaches to cell mechanical characterization generally have limited throughput, emerging microscale technologies are opening new opportunities for high-throughput deformability cytometry at the single-cell level.
Methods:
In this work, we propose an innovative microfluidic system based on (i) a hyperbolic microchannel to induce erythrocyte deformation by extensional flow, and (ii) an electrical sensing zone with coplanar electrodes to evaluate the deformed cell shape.
Results:
RBC deformation under extensional flow is achieved, and the deformed cell shape is quantified by means of an electrical anisotropy index, at a throughput of 300 cell/s. Measurements of healthy and chemically stiffened RBCs demonstrate that the anisotropy index can be used to characterize RBC deformability, as an alternative to deformation indices based on high-speed image processing.
Conclusion:
A contactless and optics-free approach for RBC deformability analysis has been presented.
Significance:
Due to its simplicity and potential for integration, the proposed approach holds promises for fast and low-cost erythrocyte deformability assays, especially in point-of-care and resource-limited settings.
... Other researchers have introduced a microfluidic with constriction channel, which is a simple and high throughput technique to determine the electrical characteristics of a single cell (e.g. specific membrane capacitance) [16][17][18][19]. However, the microfluidic device with a constriction channel approach is difficult to optimize for sizeheterogeneous samples. ...
Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution, and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially-available Tungsten needle coated with parylene. When a single cell flows through the parallel facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m ⁻² , with the regression coefficients, E at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.
... Microfluidic impedance flow cytometry (IFC) [13][14], a major branch of electrical biosensors, has emerged as a labelfree, low-cost, high-throughput method for rapid particle sizing [15] and cell analysis [16]. When cells pass through the sensing zone, the resistance between the electrodes changes accordingly, resulting in electrical pulses related to the size and other dielectric parameters of cells. ...
According to the urgent need for rapid detection and identification of foodborne bacteria to prevent public health event, a microfluidic electrical impedance flow cytometry assisted with convolutional neural network (ConvNet) based deep learning algorithm was proposed in this study to analyze the impedance signals of bacteria. With the assistance of the deep learning algorithm, Escherichia coli (EPEC), Salmonella enteritidis (SE) and Vibrio parahaemolyticus (VP) were identified with an accuracy of 100%. The proposed impedance based analysis system can be potentially applied for pre-classification of different subtypes of bacteria in a label-free manner.Clinical Relevance-The whole platform can be miniaturized and applied for point-of-care testing (POCT) of pathogenic bacteria detection.
... In these works, microfluidic chips are widely utilized for single cell capture, isolation, screening, and other cell manipulations. Many publications have reported state-of-art chips and platforms for single cell research [2,3], including bioactive molecules detection, circulating tumor cells enrichment, cytotoxicity analysis, mechanical and electrical cellular characterization. Thus, microfluidic single cell manipulation is a hot research topic and potentially powerful for lots of biomedical applications. ...
Single cell research with microfluidic chip is of vital importance in biomedical studies and clinical medicine. Simultaneous microfluidic cell manipulations and long term cell monitoring needs further investigations due to the lack of label‐free quantitative imaging techniques and systems. In this work, single cell capture, isolation and long term in‐situ monitoring was realized with a microfluidic cell chip, compact cell incubator and quantitative self‐interference spectroscopy. The proposed imaging method could obtain quantitative and dynamic refractive index distribution in living cells. And the designed microfluidic chip could capture and isolate single cells. The customized incubator could support cell growth conditions when single cell was captured in microfluidic chip. According to the results, single cells could be trapped, transferred and pushed into the culture chamber with the microfluidic chip. The incubator could culture single cells in the chip for 120 h. The refractive index sensitivity of the proposed quantitative imaging method was 0.0282 and the relative error was merely 0.04%.
Electrical impedance cytometry (EIC) serves as a crucial tool within the realm of microfluidics, providing a label-free, precise, and high-throughput means for assessing cells and particles. In this study, we explore the optimal experimental parameters in terms of EIC for droplet measurement. Initially, a microfluidic platform incorporating an EIC module is established. Within this setup, droplets comprising phosphate-buffered saline (PBS) solution are generated. They flow through the microchannel positioned above the EIC region for signal outputs. The relationships between droplet length, electrode length, and droplet interval distance are systematically investigated. Optimal conditions for achieving complete signal waveforms are identified, wherein the electrode length exceeds the droplet length but falls short of the droplet interval distance. This is corroborated by both simulation and experimental results. Furthermore, droplet geometry proves to influence the shape of the generated signal waveform. Finally, the microfluidic system demonstrates accurate measurements of droplet lengths spanning 95, 230, and
m at total flow rates of 24, 30, and
L/min, respectively. This study is anticipated to elucidate the underlying principles guiding ECI design and further contribute to advancements in the field of droplet microfluidics.
Chemotherapeutic drugs such as paclitaxel and vinblastine interact with microtubules and thus induce complex cell states of mitosis arrest at the G2/M phase followed by apoptosis dependent on drug exposure time and concentration. Microfluidic impedance cytometry (MIC), as a label-free and high-throughput technology for single-cell analysis, has been applied for viability assay of cancer cells post drug exposure at fixed time and dosage, yet verification of this technique for varied tumor cell states after anticancer drug treatment remains a challenge. Here we present a novel MIC device and for the first time perform impedance cytometry on carcinoma cells exhibiting progressive states of G2/M arrest followed by apoptosis related to drug concentration and exposure time, after treatments with paclitaxel and vinblastine, respectively. Our results from impedance cytometry reveal increased amplitude and negative phase shift at low frequency as well as higher opacity for HeLa cells under G2/M mitotic arrest compared to untreated cells. The cells under apoptosis, on the other hand, exhibit opposite changes in these electrical parameters. Therefore, the impedance features differentiate the HeLa cells under progressive states post anticancer drug treatment. We also demonstrate that vinblastine poses a more potent drug effect than paclitaxel especially at low concentrations. Our device is fabricated using a unique sacrificial layer-free soft lithography process as compared to the existing MIC device, which gives rise to readily aligned parallel microelectrodes made of silver-PDMS embedded in PDMS channel sidewalls with one molding step. Our results uncover the potential of the MIC device, with a fairly simple and low-cost fabrication process, for cellular state screening in anticancer drug therapy.
Biomicrofluidics, a subdomain of microfluidics, has been inspired by several ideas from nature. However, while the basic inspiration for the same may be drawn from the living world, the translation of all relevant essential functionalities to an artificially engineered framework does not remain trivial. Here, we review the recent progress in bio-inspired microfluidic systems via harnessing the integration of experimental and simulation tools delving into the interface of engineering and biology. Development of “on-chip” technologies as well as their multifarious applications is subsequently discussed, accompanying the relevant advancements in materials and fabrication technology. Pointers toward new directions in research, including an amalgamated fusion of data-driven modeling (such as artificial intelligence and machine learning) and physics-based paradigm, to come up with a human physiological replica on a synthetic bio-chip with due accounting of personalized features, are suggested. These are likely to facilitate physiologically replicating disease modeling on an artificially engineered biochip as well as advance drug development and screening in an expedited route with the minimization of animal and human trials.
Biological parameters extracted from electrical signals from various body parts have been used for many years to analyze the human body and its behavior. In addition, electrical signals from cancer cell lines, normal cells, and viruses, among others, have been widely used for the detection of various diseases. Single-cell parameters such as cell and cytoplasmic conductivity, relaxation frequency, and membrane capacitance are important. There are many techniques available to characterize biomaterials, such as nanotechnology, microstrip cavity resonance measurement, etc. This article reviews single-cell isolation and sorting techniques, such as the micropipette separation method, separation and sorting system (dual electrophoretic array system), DEPArray sorting system (dielectrophoretic array system), cell selector sorting system, and microfluidic and valve devices, and discusses their respective advantages and disadvantages. Furthermore, it summarizes common single-cell electrical manipulations, such as single-cell amperometry (SCA), electrical impedance sensing (EIS), impedance flow cytometry (IFC), cell-based electrical impedance (CEI), microelectromechanical systems (MEMS), and integrated microelectrode array (IMA). The article also enumerates the application and significance of single-cell electrochemical analysis from the perspectives of CTC liquid biopsy, recombinant adenovirus, tumor cells like lung cancer DTCs (LC-DTCs), and single-cell metabolomics analysis. The paper concludes with a discussion of the current limitations faced by single-cell analysis techniques along with future directions and potential application scenarios.
Cell sorting is an essential prerequisite for cell research and has great value in life science and clinical studies. Among the many microfluidic cell sorting technologies, label-free methods based on the size of different cell types have been widely studied. However, the heterogeneity in size for cells of the same type and the inevitable size overlap between different types of cells would result in performance degradation in size-based sorting. To tackle such challenges, deformation-assisted technologies are receiving more attention recently. Cell deformability is an inherent biophysical marker of cells that reflects the changes in their internal structures and physiological states. It provides additional dimensional information for cell sorting besides size. Therefore, in this review, we summarize the recent advances in deformation-assisted microfluidic cell sorting technologies. According to how the deformability is characterized and the form in which the force acts, the technologies can be divided into two categories: (1) the indirect category including transit-time-based and image-based methods, and (2) the direct category including microstructure-based and hydrodynamics-based methods. Finally, the separation performance and the application scenarios of each method, the existing challenges and future outlook are discussed. Deformation-assisted microfluidic cell sorting technologies are expected to realize greater potential in the label-free analysis of cells.
Background:
Somatic cell fusion is a process that transfers cytoplasmic and nuclear genes to create new germplasm resources. But our limited understanding of the physiological and molecular mechanisms that shape protoplast responses to fusion.
Method:
We employed flow cytometry, cytology, proteomics, and gene expression analysis to examine the sugarcane (Saccharum spp.) protoplast fusion.
Results:
Flow cytometry analysis revealed the fusion rate of protoplasts was 1.95%, the FSC value and SSC of heterozygous cells was 1.17-1.47 times higher than that of protoplasts. The protoplasts viability decreased and the MDA increased after fusion. During fusion, the cell membranes were perforated to different degrees, nuclear activity was weakened, while microtubules depolymerized and formed several short rod like structures in the protoplasts. The most abundant proteins during fusion were mainly involved in RNA processing and modification, cell cycle control, cell division, chromosome partition, nuclear structure, extracellular structures, and nucleotide transport and metabolism. Moreover, the expression of key regeneration genes, such as WUS, GAUT, CESA, PSK, Aux/IAA, Cdc2, Cyclin D3, Cyclin A, and Cyclin B, was significantly altered following fusion.
Purpose and significance:
Overall, our findings provide a theoretical basis that increases our knowledge of the mechanisms underlying protoplast fusion.
As a label-free single cell analysis approach, impedance flow cytometry provides valuable information by differential signals. However, for high throughput purpose, particle coincidence is inevitable especially when the cell concentration or pumping rate is too high. In this work, we proposed the first numerical model and a novel method for particle coincidence. To investigate the multi-particle events, a double differential impedance sensor is used. From a statistic analysis of signal peaks, we could determine the threshold of particle events, and then read out particle numbers. Compared with customized threshold, the data-driven cut-off line fundamentally improves the accuracy of particle recognition. Herein, the key operation parameters that influence the particle coincidence including sample concentration, driven pressure (stands for pumping rate), and sensor configuration are elucidated. Moreover, a guide to the best practices for avoid multi-particle coincidence events are suggested for real applications.
Microfluidic cytometry (MC) and electrical impedance spectroscopy (EIS) are two important techniques in biomedical engineering. Microfluidic cytometry has been utilized in various fields such as stem cell differentiation and cancer metastasis studies, and provides a simple, label-free, real-time method for characterizing and monitoring cellular fates. The impedance microdevice, including impedance flow cytometry (IFC) and electrical impedance spectroscopy (EIS), is integrated into MC systems. IFC measures the impedance of individual cells as they flow through a microfluidic device, while EIS measures impedance changes during binding events on electrode regions. There have been significant efforts to improve and optimize these devices for both basic research and clinical applications, based on the concepts, electrode configurations, and cell fates. This review outlines the theoretical concepts, electrode engineering, and data analytics of these devices, and highlights future directions for development.
This study presents a microfluidic impedance flow cytometry enabling the quantification of intrinsic single-cell bio-dielectric parameters (e.g., cell radius of
, relative membrane permittivity of
, and cytoplasmic conductivity of
) based on constrictional microchannel, numerical simulation, and neural network. Numerical simulation of a traveling cell within constrictional microchannel was conducted to locate relationship between intrinsic bio-dielectric parameters of single cells with corresponding impedance profiles, functioning as “reference” cells for the feedforward neural network, producing fitting accuracies of 1.00 ± 0.00 of
, 1.00 ± 0.00 of
, and 1.00 ± 0.01 of
. Then, the fitted analytical equation based on numerical simulation and neural network was further used to translate measured impedance profiles into intrinsic bio-dielectric parameters of single cells, where
,
, and
were quantified as 7.6 ±
, 16.73 ± 2.97, and 1.38 ± 0.34 S/m of K562 cells (
), 6.4 ±
, 17.47 ± 5.09, and 1.46 ± 0.47 S/m of Jurkat cells (
), 5.9 ±
, 17.40 ± 3.14, and 1.96 ± 0.81 S/m of HL-60 cells (
), respectively. Compared to phenomenal electrical signals (i.e., real and imaginary parts at specific frequency) detected by conventional electrical flow cytometry, which were prone to environmental variations, this microfluidic platform enabled the quantification of intrinsic bio-dielectric parameters of single cells and thus provides an effective tool in the field of flow cytometry.
Targeted delivery and labeling of single living cells in heterogeneous cell populations are of great importance to understand the molecular biology and physiological functions of individual cells. However, it remains challenging to perfuse fluorescence markers into single living cells with high spatial and temporal resolution without interfering neighboring cells. Here, we report a single cell perfusion and fluorescence labeling strategy based on nanoscale glass nanopipettes. With the nanoscale tip hole of 100 nm, the use of nanopipettes allows special perfusion and high-resolution fluorescence labeling of different subcellular regions in single cells of interest. The dynamic of various fluorescent probes has been studied to exemplify the feasibility of nanopipette-dependent targeted delivery. According to experimental results, the cytoplasm labeling of Sulfo-Cyanine5 and fluorescein isothiocyanate is mainly based on the Brownian movement due to the dyes themselves and does not have a targeting ability, while the nucleus labeling of 4',6-diamidino-2-phenylindole (DAPI) is originated from the adsorption between DAPI and DNA in the nucleus. From the finite element simulation, the precise manipulation of intracellular delivery is realized by controlling the electro-osmotic flow inside the nanopipettes, and the different delivery modes between nontargeting dyes and nucleus-targeting dyes were compared, showcasing the valuable ability of nanopipette-based method for the analysis of specially defined subcellular regions and the potential applications for single cell surgery, subcellular manipulation, and gene delivery.
Accurate analysis of the mechanical, electrical, and optical properties of single cells can provide useful information on their physiological and pathological states. Microfluidics has become a promising tool for single-cell analysis because it greatly reduces the operation complexity, reduces the reagent consumption, and enables the integration of multiple functions. In recent years, the rapid development of microfluidic detection technologies is laying a foundation for single-cell analysis, which promotes the in-depth analysis and understanding of cells. In this review, we provide a comprehensive overview of the most recent advances on label-free microfluidics for analyzing the biophysical properties of single cells. Herein, we present the applications of cell biophysical properties for identifying cell types, distinguishing between healthy and diseased cells, and studying the drug resistance of cells. Lastly, the advantages, limitations, and technical characteristics of each method are discussed.
Single-cell analysis has gained considerable attention for disease diagnosis, drug screening, and differentiation monitoring. Compared to the well-established flow cytometry, which uses fluorescent-labeled antibodies, microfluidic impedance cytometry (MIC) offers a simple, label-free, and noninvasive method for counting, classifying, and monitoring cells. Superior features including a small footprint, low reagent consumption, and ease of use have also been reported. The MIC device detects changes in the impedance signal caused by cells passing through the sensing/electric field zone, which can extract information regarding the size, shape, and dielectric properties of these cells. According to recent studies, electrode configuration has a remarkable effect on detection accuracy, sensitivity, and throughput. With the improvement in microfabrication technology, various electrode configurations have been reported for improving detection accuracy and throughput. However, the various electrode configurations of MIC devices have not been reviewed. In this review, the theoretical background of the impedance technique for single-cell analysis is introduced. Then, two-dimensional, three-dimensional, and liquid electrode configurations are discussed separately; their sensing mechanisms, fabrication processes, advantages, disadvantages, and applications are also described in detail. Finally, the current limitations and future perspectives of these electrode configurations are summarized. The main aim of this review is to offer a guide for researchers on the ongoing advancement in electrode configuration designs.
Analysis on a single-cell basis is both fundamental and meaningful in biomedical research and clinical practice. Flow cytometry is one of the most popular approaches in this field with broad applications in cell sorting, counting, and identification of rare cells. However, the complicated design and bulky size of conventional flow cytometry have restricted their applications mainly in centralized laboratories. With the recent development of smartphone devices, smartphone-based cytometry has been explored and tested for single-cell analysis. Compared with traditional cytometers, smartphone-based cytometric biosensors are more suitable for point-of-care (POC) uses, such as on-site disease diagnosis and personal health monitoring. In this review article, the history of traditional flow cytometry is introduced, and advances of smartphone-enabled cytometry are summarized in detail based on different working principles. Representative POC applications of smartphone cytometers are also discussed. The achievements demonstrated so far illustrate the potential of smartphone-based cytometric devices to transform single-cell measurement in general, with a significant impact in POC diagnostics, preventive medicine, and cell biology. Keywords: Flow cytometry, Smartphone, Point-of-care diagnostics, Single-cell analysis, Imaging, Microfluidics
The life cycle and development of plants requires the biosynthesis, deposition, and degradation of cell wall matrix polysaccharides. The structures of the diverse cell wall matrix polysaccharides influence commercially important properties of plant cells, including growth, biomass recalcitrance, organ abscission, and the shelf life of fruits. This review is a comprehensive summary of the matrix polysaccharide glycosyltransferase (GT) activities that have been verified using in vitro assays following heterologous GT protein expression. Plant cell wall (PCW) biosynthetic GTs are primarily integral transmembrane proteins localized to the endoplasmic reticulum and Golgi of the plant secretory system. The low abundance of these enzymes in plant tissues makes them particularly difficult to purify from native plant membranes in quantities sufficient for enzymatic characterization, which is essential to study the functions of the different GTs. Numerous activities in the synthesis of the major cell wall matrix glycans, including pectins, xylans, xyloglucan, mannans, mixed-linkage glucans (MLGs), and arabinogalactan components of AGP proteoglycans have been mapped to specific genes and multi-gene families. Cell wall GTs include those that synthesize the polymer backbones, those that elongate side branches with extended glycosyl chains, and those that add single monosaccharide linkages onto polysaccharide backbones and/or side branches. Three main strategies have been used to identify genes encoding GTs that synthesize cell wall linkages: analysis of membrane fractions enriched for cell wall biosynthetic activities, mutational genetics approaches investigating cell wall compositional phenotypes, and omics-directed identification of putative GTs from sequenced plant genomes. Here we compare the heterologous expression systems used to produce, purify, and study the enzyme activities of PCW GTs, with an emphasis on the eukaryotic systems Nicotiana benthamiana, Pichia pastoris, and human embryonic kidney (HEK293) cells. We discuss the enzymatic properties of GTs including kinetic rates, the chain lengths of polysaccharide products, acceptor oligosaccharide preferences, elongation mechanisms for the synthesis of long-chain polymers, and the formation of GT complexes. Future directions in the study of matrix polysaccharide biosynthesis are proposed.
The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.
Individual cells in an organism are variable, which strongly impacts cellular processes. Advances in sequencing technologies have enabled single-cell genomic analysis to become widespread, addressing shortcomings of analyses conducted on populations of bulk cells. While the field of single-cell plant genomics is in its infancy, there is great potential to gain insights into cell lineage and functional cell types to help understand complex cellular interactions in plants. In this review, we discuss current approaches for single-cell plant genomic analysis, with a focus on single-cell isolation, DNA amplification, next-generation sequencing, and bioinformatics analysis. We outline the technical challenges of analysing material from a single plant cell, and then examine applications of single-cell genomics and the integration of this approach with genome editing. Finally, we indicate future directions we expect in the rapidly developing field of plant single-cell genomic analysis.
How complex developmental-genetic networks are translated into organs with specific 3D shapes remains an open question. This question is particularly challenging because the elaboration of specific shapes is in essence a question of mechanics. In plants, this means how the genetic circuitry affects the cell wall. The mechanical properties of the wall and their spatial variation are the key factors controlling morphogenesis in plants. However, these properties are difficult to measure and investigating their relation to genetic regulation is particularly challenging. To measure spatial variation of mechanical properties, one must determine the deformation of a tissue in response to a known force with cellular resolution. Here we present an automated confocal micro-extensometer (ACME), which greatly expands the scope of existing methods for measuring mechanical properties. Unlike classical extensometers, ACME is mounted on a confocal microscope and utilizes confocal images to compute the deformation of the tissue directly from biological markers, thus providing 3D cellular scale information and improved accuracy. Additionally, ACME is suitable for measuring the mechanical responses in live tissue. As a proof of concept, we demonstrate that the plant hormone gibberellic acid induces a spatial gradient in mechanical properties along the length of the Arabidopsis hypocotyl.
At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.
Microfluidic impedance cytometry offers a simple non-invasive method for single-cell analysis. Coplanar electrode chips are especially attractive due to ease of fabrication, yielding miniaturized, reproducible, and ultimately low-cost devices. However, their accuracy is challenged by the dependence of the measured signal on particle trajectory within the interrogation volume, that manifests itself as an error in the estimated particle size, unless any kind of focusing system is used. In this paper, we present an original five-electrode coplanar chip enabling accurate particle sizing without the need for focusing. The chip layout is designed to provide a peculiar signal shape from which a new metric correlating with particle trajectory can be extracted. This metric is exploited to correct the estimated size of polystyrene beads of 5.2, 6 and 7 μm nominal diameter, reaching coefficient of variations lower than the manufacturers' quoted values. The potential impact of the proposed device in the field of life sciences is demonstrated with an application to Saccharomyces cerevisiae yeast.
Growth in plants results from the interaction between genetic and signalling networks and the mechanical properties of cells and tissues. There has been a recent resurgence in research directed at understanding the mechanical aspects of growth, and their feedback on genetic regulation. This has been driven in part by the development of new micro-indentation techniques to measure the mechanical properties of plant cells in vivo. However, the interpretation of indentation experiments remains a challenge, since the force measures results from a combination of turgor pressure, cell wall stiffness, and cell and indenter geometry. In order to interpret the measurements, an accurate mechanical model of the experiment is required. Here, we used a plant cell system with a simple geometry, Nicotiana tabacum Bright Yellow-2 (BY-2) cells, to examine the sensitivity of micro-indentation to a variety of mechanical and experimental parameters. Using a finite-element mechanical model, we found that, for indentations of a few microns on turgid cells, the measurements were mostly sensitive to turgor pressure and the radius of the cell, and not to the exact indenter shape or elastic properties of the cell wall. By complementing indentation experiments with osmotic experiments to measure the elastic strain in turgid cells, we could fit the model to both turgor pressure and cell wall elasticity. This allowed us to interpret apparent stiffness values in terms of meaningful physical parameters that are relevant for morphogenesis.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions.
Morphogenesis, the remarkable process by which a developing organism achieves its shape, relies on the coordinated growth
of cells, tissues, and organs. While the molecular and genetic basis of morphogenesis is starting to be unravelled, understanding
shape changes is lagging behind. Actually, shape is imposed by the structural elements of the organism, and the translation
of cellular activity into morphogenesis must go through these elements. Therefore, many methods have been developed recently
to quantify, at cellular resolution, the properties of the main structural element in plants, the cell wall. As plant cell
growth is restrained by the cell wall and powered by turgor pressure, such methods also address the quantification of turgor.
These different micromechanical approaches are reviewed here, with a critical assessment of their strengths and weaknesses,
and a discussion of how they can help us understand the regulation of growth and morphogenesis.
Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.
Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall.
The effect of salinity on cell turgor, plasma membrane permeability and cell wall elasticity has been measured in petioles of an aspen hybrid using the cell pressure probe. Control plants were grown in soil without the addition of NaCl and treated plants were grown in soil with 50 mM of NaCl for 1, 2, 3 and 4 weeks. In parenchyma cells from Populus tremula × tremuloides petioles with an increased level of NaCl in the soil: (a) turgor pressure was reduced after 1 week of treatment but afterward it was similar to untreated plants, (b) the value of elastic modulus of the cell walls increased, and (c) hydraulic conductivity of the plasma membrane of treated plants decreased in comparison to untreated ones. No histological differences and distribution of JIM5 antibody between the petioles of plants grown under salinity and the untreated were found. In cell walls of parenchyma and collenchyma from plants grown under salinity, the presence of pectic epitopes recognized by JIM7 antibodies was increased in comparison to the control plants. The obtained results indicate that under salt stress the permeability of water through plasma membrane is disturbed, cell walls became more rigid but the turgor pressure did not change.
Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.
This paper presents a microfluidic system for cell type classification using mechanical and electrical measurements on single cells. Cells are aspirated continuously through a constriction channel with cell elongations and impedance profiles measured simultaneously. The cell transit time through the constriction channel and the impedance amplitude ratio are quantified as cell's mechanical and electrical property indicators. The microfluidic device and measurement system were used to characterize osteoblasts (n=206) and osteocytes (n=217), revealing that osteoblasts, compared with osteocytes, have a larger cell elongation length (64.51 ± 14.98 μm vs. 39.78 ± 7.16 μm), a longer transit time (1.84 ± 1.48 s vs. 0.94 ± 1.07 s), and a higher impedance amplitude ratio (1.198 ± 0.071 vs. 1.099 ± 0.038). Pattern recognition using the neural network was applied to cell type classification, resulting in classification success rates of 69.8% (transit time alone), 85.3% (impedance amplitude ratio alone), and 93.7% (both transit time and impedance amplitude ratio as input to neural network) for osteoblasts and osteocytes. The system was also applied to test EMT6 (n=747) and EMT6/AR1.0 cells (n=770, EMT6 treated by doxorubicin) that have a comparable size distribution (cell elongation length: 51.47 ± 11.33 μm vs. 50.09 ± 9.70 μm). The effects of cell size on transit time and impedance amplitude ratio were investigated. Cell classification success rates were 51.3% (cell elongation alone), 57.5% (transit time alone), 59.6% (impedance amplitude ratio alone), and 70.2% (both transit time and impedance amplitude ratio). These preliminary results suggest that biomechanical and bioelectrical parameters, when used in combination, could provide a higher cell classification success rate than using electrical or mechanical parameter alone.
Higher plants resist the forces of gravity and powerful lateral forces through the cumulative strength of the walls that surround individual cells. These walls consist mainly of cellulose, noncellulosic polysaccharides and lignin, in proportions that depend upon the specific functions of the cell and its stage of development. Spatially and temporally controlled heterogeneity in the physicochemical properties of wall polysaccharides is observed at the tissue and individual cell levels, and emerging in situ technologies are providing evidence that this heterogeneity also occurs across a single cell wall. We consider the origins of cell wall heterogeneity and identify contributing factors that are inherent in the molecular mechanisms of polysaccharide biosynthesis and are crucial for the changing biological functions of the wall during growth and development. We propose several key questions to be addressed in cell wall biology, together with an alternative two-phase model for the assembly of noncellulosic polysaccharides in plants.
In the arbuscular mycorrhizal symbiosis, the fungal symbiont colonizes root cortical cells, where it establishes differentiated hyphae called arbuscules. As each arbuscule develops, the cortical cell undergoes a transient reorganization and envelops the arbuscule in a novel symbiosis-specific membrane, called the periarbuscular membrane. The periarbuscular membrane, which is continuous with the plant plasma membrane of the cortical cell, is a key interface in the symbiosis; however, relatively little is known of its composition or the mechanisms of its development. Here, we used fluorescent protein fusions to obtain both spatial and temporal information about the protein composition of the periarbuscular membrane. The data indicate that the periarbuscular membrane is composed of at least two distinct domains, an "arbuscule branch domain" that contains the symbiosis-specific phosphate transporter, MtPT4, and an "arbuscule trunk domain" that contains MtBcp1. This suggests a developmental transition from plasma membrane to periarbuscular membrane, with biogenesis of a novel membrane domain associated with the repeated dichotomous branching of the hyphae. Additionally, we took advantage of available organelle-specific fluorescent marker proteins to further evaluate cells during arbuscule development and degeneration. The three-dimensional data provide new insights into relocation of Golgi and peroxisomes and also illustrate that cells with arbuscules can retain a large continuous vacuolar system throughout development.
We propose a model to determine the influence of different cell properties, such as size, membrane capacitance and cytoplasm conductivity, on the impedance spectrum as measured in a microfabricated cytometer. A dielectric sphere of equivalent complex permittivity is used as a simplified model to describe a biological cell. The measurement takes place between a pair of facing microelectrodes in a microchannel filled with a saline solution. The model incorporates various cell parameters, such as dielectric properties, size and position in the channel. A 3D finite element model is used to evaluate the magnitude of the electric field in the channel and the resultant changes in charge densities at the measurement electrode boundaries as a cell flows past. The charge density is integrated on the electrode surface to determine the displacement current and the channel impedance for the computed frequency range. The complete impedance model combines the finite element model, the electrode-electrolyte interface impedance and stray impedance, which are measured from a real device. The modeled dielectric complex spectra for various cell parameters are discussed and a measurement strategy for cell discrimination with such a system is proposed. We finally discuss the amount of noise and measurement fluctuations of the sensor.
One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses
of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding
of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are
controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into
a useful model will require a system-based approach.
Primary cell wall (PCW) is a rigid yet flexible cell wall surrounding plant cells and it plays key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. As a technique widely used to study the characteristics of mammalian cells, electrical impedance spectroscopy (EIS) is rarely used in plant science. In this work, we designed and fabricated an EIS based biosensor coupled with microfluidic platform to investigate the formation process of primary cell wall (PCW) at the single-cell level. Arabidopsis mesophyll cells with completely regenerated PCW showed significantly higher impedance values compared to the nascent protoplasts without PCW, demonstrating that PCW formation caused a dramatic change in cell electrical properties. The device could also discriminate plant mutant cells with modified PCW compositions, thus provided a novel tool for physical phenotyping of plant cells. The dose-dependent effects of exogenously applied auxin on PCW regeneration were corroborated on this platform which revealed its potential to sensitively detect the influences of in vitro stimuli. This work not only provided one novel application of impedance-based biosensor to characterize a plant-specific developmental event, but also revealed the promises of EIS integrated microfluidic system as a sensitive, time-effective and low-cost platform to characterize single plant cells and make new scientific discoveries in plant science.
The detection of circulating tumor cells (CTCs) from liquid biopsies using microfluidic devices is attracting a considerable amount of attention as a new, less-invasive cancer diagnostic and prognostic method. One of the drawbacks of the existing antibody-based detection systems is the false negatives for epithelial cell adhesion molecule detection of CTCs. Here, we report a mechanical low-pass filtering technique based on a microfluidic constriction and electrical current sensing system for the novel CTC detection in whole blood without any specific antigen-antibody interaction or biochemical modification of the cell surface. The mechanical response of model cells of CTCs, such as HeLa, A549, and MDA-MB-231 cells clearly demonstrated different behaviors from that of Jurkat cells, a human T-lymphocyte cell line, when they passed through the 6-μm wide constriction channel. A 6-μm wide constriction channel was determined as the optimum size to identify CTCs in whole blood with an accuracy greater than 95% in tens of milliseconds. The mechanical filtering of cells at a single cell level was achieved from a whole blood without any pretreatment (e.g. dilution of lysing) and pre-labeling (e.g. fluorophores or antibodies).
Precise measurement of mechanical and electrical properties of single cells can yield useful information on the physiological and pathological state of cells. In this work, we develop a differential multiconstriction microfluidic device with self-aligned 3D electrodes to simultaneously characterize the deformability, electrical impedance and relaxation index of single cells at a high throughput manner (>430 cell/min). Cells are pressure-driven to flow through a series of sequential microfluidic constrictions, during which deformability, electrical impedance and relaxation index of single cells are extracted simultaneously from impedance spectroscopy measurements. Mechanical and electrical phenotyping of untreated, Cytochalasin B treated and N-Ethylmaleimide treated MCF-7 breast cancer cells demonstrate the ability of our system to distinguish different cell populations purely based on these biophysical properties. In addition, we quantify the classification of different cell types using a back propagation neural network. The trained neural network yields the classification accuracy of 87.8% (electrical impedance), 70.1% (deformability), 42.7% (relaxation index) and 93.3% (combination of electrical impedance, deformability and relaxation index) with high sensitivity (93.3%) and specificity (93.3%) for the test group. Furthermore, we have demonstrated the cell classification of a cell mixture using the presented biophysical phenotyping technique with the trained neural network, which is in quantitative agreement with the flow cytometric analysis using fluorescent labels. The developed concurrent electrical and mechanical phenotyping provide great potential for high-throughput and label-free single cell analysis.
While plant growth is well known to rely on turgor pressure, it is challenging to quantify the contribution of turgor pressure to plant cell rheology. Here we used a custom-made micro-rheometer to quantify the viscoelastic behavior of isolated plant cells while varying their internal turgor pressure. To get insight into how plant cells adapt their internal pressure to the osmolarity of their medium, we compared the mechanical behavior of single plant cells to that of a simple, passive, pressurized shell: a soccer ball. While both systems exhibited the same qualitative behavior, a simple mechanical model allowed us to quantify turgor pressure regulation at the single cell scale.
As stem cell therapies become more common in the clinic, there is a greater need for real-time, label-free monitoring of the differentiation status of the cells. In this paper, we present a dual-micropore-based, high-throughput microfluidic electrical impedance flow cytometer for non-invasive identification of the differentiation state of mesenchymal stem cells. The mesenchymal stem cells were induced to differentiate into osteoblasts over a 21 day period. Samples of mesenchymal stem cells and osteoblasts were flowed through the device, and impedance measurements were acquired over a frequency range from 50 kHz to 10 MHz. The opacity and relative angle, which shed light on the membrane capacitance and interior dielectric properties of cells, were used as interrogation parameters to analyze collected impedance data. Specifically, identification of mesenchymal stem cells and osteoblasts in a mixed population was optimized using a combination of opacity signature at 500 kHz and relative angle at 3 MHz. Identification of both cell populations in a mixed sample was successfully achieved with an accuracy of 87%. The results show a progressive increase in the number of osteoblasts throughout the 21 day differentiation process, with 36% more mesenchymal stem cells differentiated after 14 days of induction compared to after just 7 days. The dual-micropore microfluidic impedance flow cytometer system may become an important non-invasive tool for assessing stem cell quality and differentiation stages for future regenerative medicine applications.
We developed a microfluidic sensor for label-free flow cytometric cell differentiation by combined multiple AC electrical impedance and light scattering analysis. The measured signals are correlated to cell volume, membrane capacity and optical properties of single cells. For an improved signal to noise ratio, the microfluidic sensor incorporates two electrode pairs for differential impedance detection. One-dimensional sheath flow focusing was implemented, which allows single particle analysis at kHz count rates. Various monodisperse particles and differentiation of leukocytes in haemolysed samples served to benchmark the microdevice applying combined AC impedance and side scatter analyses. In what follows, we demonstrate that AC impedance measurements at selected frequencies allow label-free discrimination of platelets, erythrocytes, monocytes, granulocytes and lymphocytes in whole blood samples involving dilution only. Immunofluorescence staining was applied to validate the results of the label-free cell analysis. Reliable differentiation and enumeration of cells in whole blood by AC impedance detection have the potential to support medical diagnosis for patients with haemolysis resistant erythrocytes or abnormally sensitive leucocytes, i.e. for patients suffering from anaemia or leukaemia.
The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.
The dielectric properties of tumour
cells are known to differ from normal blood cells, and this difference can be exploited for label-free separation of cells. Conventional measurement techniques are slow and cannot identify rare circulating tumour
cells (CTCs) in a realistic timeframe. We use high throughput single cell
microfluidic impedance cytometry to measure the dielectric properties of the MCF7 tumour
cell line (representative of CTCs), both as pure populations and mixed with whole blood. The data show that the MCF7 cells have a large membrane
capacitance and size, enabling clear discrimination from all other leukocytes. Impedance analysis is used to follow changes in cell viability when cells are kept in suspension, a process which can be understood from modelling time-dependent changes in the dielectric properties (predominantly membrane conductivity) of the cells. Impedance cytometry is used to enumerate low numbers of MCF7 cells spiked into whole blood. Chemical lysis is commonly used to remove the abundant erythrocytes, and it is shown that this process does not alter the MCF7 cell count or change their dielectric properties. Combining impedance cytometry with magnetic bead based antibody enrichment enables MCF7 cells to be detected down to 100 MCF7 cells in 1 ml whole blood, a log 3.5 enrichment and a mean recovery of 92%. Microfluidic impedance cytometry could be easily integrated within complex cell separation systems for identification and enumeration of specific cell types, providing a fast in-line single cell characterisation method.
We present a novel Multi-Regime Analysis (MRA) routine for interpreting force indentation measurements of soft materials using atomic force microscopy. The MRA approach combines both well established and semi-empirical theories of contact mechanics within a single framework to deconvolute highly complex and non-linear force-indentation curves. The fundamental assumption in the present form of the model is that each structural contribution to the mechanical response acts in series with other ‘mechanical resistors’. This simplification enables interpretation of the micromechanical properties of materials with hierarchical structures and it allows automated processing of large data sets, which is particularly indispensable for biological systems. We validate the algorithm by demonstrating for the first time that the elastic modulus of polydimethylsiloxane (PDMS) films is accurately predicted from both approach and retraction branches of force-indentation curves. For biological systems with complex hierarchical structures, we show the unique capability of MRA to map the micromechanics of live plant cells, revealing an intricate sequence of mechanical deformations resolved with precision that is unattainable using conventional methods of analysis. We recommend the routine use of MRA to interpret AFM force-indentation measurements for other complex soft materials including mammalian cells, bacteria and nanomaterials.
Plant and animals have evolved different strategies for their development. Whether this is linked to major differ-ences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.
Conventional methods of plant cell analysis rely on growing plant cells in soil pots or agarose plates, followed by screening the plant phenotypes in traditional greenhouses and growth chambers. These methods are usually costly, need a large number of experiments, suffer from low spatial resolution and disorderly growth behavior of plant cells, with lack of ability to locally and accurately manipulate the plant cells. Microfluidic platforms take advantage of miniaturization for handling small volume of liquids and providing a closed environment, with the purpose of in vitro single cell analysis and characterizing cell response to external cues. These platforms have shown their ability for high-throughput cellular analysis with increased accuracy of experiments, reduced cost and experimental times, versatility in design, ability for large-scale and combinatorial screening, and integration with other miniaturized sensors. Despite extensive research on animal cells within microfluidic environments for high-throughput sorting, manipulation and phenotyping studies, the application of microfluidics for plant cells studies has not been accomplished yet. Novel devices such as RootChip, RootArray, TipChip, and PlantChip developed for plant cells analysis, with high spatial resolution on a micrometer scale mimicking the internal microenvironment of plant cells, offering preliminary results on the capability of microfluidics to conquer the constraints of conventional methods. These devices have been used to study different aspects of plant cell biology such as gene expression, cell biomechanics, cellular mechanism of growth, cell division, and cells fusion. This review emphasizes the advantages of current microfluidic systems for plant science studies, and discusses future prospects of microfluidic platforms for characterizing plant cells response to diverse external cues.
A method is described for purifying plant protoplasts from cellular and subcellular debris. The procedure utilizes a density buffer containing 9.6% sodium metrizoate and 5.6% Ficoll. The use of fluorescein diacetate for assessing the viability of plant protoplasts is also reported.
Tip-growing cells have the unique property of invading living tissues and abiotic growth matrices. To do so, they exert significant penetrative forces. In plant and fungal cells, these forces are generated by the hydrostatic turgor pressure. Using the TipChip, a microfluidic lab-on-a-chip device developed for tip-growing cells, we tested the ability to exert penetrative forces generated in pollen tubes, the fastest-growing plant cells. The tubes were guided to grow through microscopic gaps made of elastic polydimethylsiloxane material. Based on the deformation of the gaps, the force exerted by the elongating tubes to permit passage was determined using finite element methods. The data revealed that increasing mechanical impedance was met by the pollen tubes through modulation of the cell wall compliance and, thus, a change in the force acting on the obstacle. Tubes that successfully passed a narrow gap frequently burst, raising questions about the sperm discharge mechanism in the flowering plants.
Biomechanical and mathematical modeling of plant developmental processes requires quantitative information about the structural and mechanical properties of living cells, tissues and cellular components. A crucial mechanical property of plant cells is the mechanical stiffness or Young's modulus of its cell wall. Measuring this property in situ at single cell wall level is technically challenging. Here, a bending test is implemented in a chip, called Bending-Lab-On-a-Chip (BLOC), to quantify this biomechanical property for a widely investigated cellular model system, the pollen tube. Pollen along with culture medium is introduced into a microfluidic chip and the growing pollen tube is exposed to a bending force created through fluid loading. The flexural rigidity of the pollen tube and the Young's modulus of the cell wall are estimated through finite element modeling of the observed fluid-structure interaction. An average value of 350 MPa was experimentally estimated for the Young's modulus in longitudinal direction of the cell wall of Camellia pollen tubes. This value is in agreement with the result of an independent method based on cellular shrinkage after plasmolysis and with the mechanical properties of in vitro reconstituted cellulose-callose material.
Large scale phenotyping of tip growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high throughput execution of more sophisticated assays, an experimental platform was developed based on microfluidic and MEMS (microelectromechanical systems) technology, the TipChip. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels harboring microscopic experimental setups. The tip growing cells, pollen tubes, filamentous yeast or fungal hyphae, can be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform we were able to answer several outstanding questions on pollen tube growth. We established that unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air raising the question how and where water is taken up by the cell. The platform opens new avenues for both, more efficient experimentation and large scale phenotyping of tip growing cells under precisely controlled, reproducible conditions. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
The phytohormone auxin is a primary regulator of growth and developmental pattern formation in plants. Auxin accumulates at specific sites (e.g., organ primordia) and induces localized growth within a tissue. Auxin also mediates developmental responses to intrinsic and external physical stimuli; however, exactly how mechanics influences auxin distribution is unknown.
Here we show that mechanical strain can regulate auxin transport and accumulation in the tomato shoot apex, where new leaves emerge and rapidly grow. Modification of turgor pressure, application of external force, and artificial growth induction collectively show that the amount and intracellular localization of the auxin efflux carrier PIN1 are sensitive to mechanical alterations. In general, the more strained the tissue was, the more PIN1 was present per cell and the higher the proportion localized to the plasma membrane. Modulation of the membrane properties alone was sufficient to explain most of the mechanical effects.
Our experiments support the hypothesis that the plasma membrane acts as a sensor of tissue mechanics that translates the cell wall strain into cellular responses, such as the intracellular localization of membrane-embedded proteins. One implication of this fundamental mechanism is the mechanical enhancement of auxin-mediated growth in young organ primordia. We propose that growth-induced mechanical strain upregulates PIN1 function and auxin accumulation, thereby promoting further growth, in a robust positive feedback loop.
Mechanical properties of cells have been shown to have a significant role in disease, as in many instances cell stiffness changes when a cell is no longer healthy. We present a high-throughput microfluidics-based approach that exploits the connection between travel time of a cell through a narrow passage and cell stiffness. The system resolves both cell travel time and relative cell diameter while retaining information on the cell level. We show that stiffer cells have longer transit times than less stiff ones and that cell size significantly influences travel times. Experiments with untreated HeLa cells and cells made compliant with latrunculin A and cytochalasin B further demonstrate that travel time is influenced by cell stiffness, with the compliant cells having faster transit time.
1.
Sections of auxin-starved hypocotyls of Helianthus annuus do not show any significant growth rate in water of buffers of pH\>-6. However, in buffers with pH-values of approximately 4, elongation growth is observed; its rate is similar to the rate of auxin-stimulated growth (after 6 h incubation). \3- This phenomenon of acid-induced growth occurs also under anaerobic conditions in contrast to auxin-induced growth (Hager 1962).
2.
Intact cell wall aggregates of Helianthus hypocotyls were obtained by complete plasmolysis of hypocotyls in 50% glycerol; cell wall associated enzymes were still active after this treatment, at least in part. While cell walls in solutions of pH\>-6 show only a small plastic extension during the first minute in response to a 50 g stretching force, a constant rate of elongation over longer periods of time (measured up to 1 h) is observed in weakly acid buffers. The highest rate of elongation is observed at about pH 4.
This acid-induced plastic extension is completely inhibited by Cu2+-ions (5mM); the elongation of cell walls is apparently the consequence of an enzyme-catalysed increase in plasticity having a pH optimum of about 4.
The pH optimum of acid-induced cell wall extension observed during stretching of plasmolysed hypocotyls coincides with the pH optimum of acid-induced growth of intact hypocotyl sections (around pH 4).
3.
Under anaerobic conditions the growth rate of intact coleoptiles stays unchanged (at a low value) if the sections are incubated in a buffer of pH 5.0. Higher proton concentrations, however, stimulate growth immediately, whereas low proton concentrations are inhibitory (Fig. 7 and 8). The strongest initial growth response is elicited by buffers or acids of pH 3.9 (Fig. 9). Acid-induced growth of coleoptiles with a similar pH optimum is also found under anaerobic conditions.
The growth of coleoptile cylinders can be switched on or off by repeatedly changing to acid or basic medium, respectively (under conditions of anaerobiosis) (Fig. 10).
IAA-induced growth (aerobic conditions, pH5) can also be inhibited immediately by basic buffers or NaOH-solutions, and resumes after the pH value is lowered (Fig. 11). This pH-dependency may be taken as an indication that auxin affects the same reaction which is stimulated by high proton concentrations and which may be the last step in the process of cell elongation.
CCCP, known to make membranes permeable for protons, rapidly inhibits the auxin-induced elongation growth (pH 6,5) when applied at a concentration which does not influence respiration; removal of CCCP shows that the growth inhibition by CCCp is partly reversible (Fig. 12). In contrast, acid-induced elongation growth (pH 4) shows inhibition by CCCP not before 10 min after application.—These findings suggest that auxin induces a proton accumulation in a cell wall compartment and, as a consequence, enzymatic cell wall softening. Such an accumulation could be brought about by an auxin-activated, membrane-bound, anisotropic ATPase or ion pump.
The notion that ATPases or pumps may be located in the outer layers of the cell membrane is supported by the observation that addition of ATP to coleoptile cylinders under anaerobic conditions results in an immediate stimulation of elongation (Fig. 14). This effect is further enhanced by Mg2+-and K+-ions (Figs. 15 and 16). Mg2+ can be partly replaced by Ca2+. The stimulatory effect of ATP is increased considerably if the coleoptiles are treated with IAA under aerobic conditions prior to ATP addition (Figs. 15 b and 14). ITP, GTP, UTP, and CTP induce elongation growth under anaerobiosis similarly to ATP. In the presence of ITP or GTP the increase in growth rate is maintained over a longer period of time than in the presence of the other nucleoside triphosphates (Fig. 17).
IAA, which causes no elongation growth under anaerobiosis (Fig. 13) is also unable to further stimulate the elongation growth induced by ATP, UTP, or CTP under anaerobiosis (Fig. 18); however, if IAA is added after growth has been stimulated by GTP or ITP, a temporary inhibition and, 10 min later, a strong stimulation is noticed (Fig. 19). If the sequence of addition is reversed, —that is, if IAA (without growth effect) and, after 20 min, GTP or ITP are added to the coleoptiles—, the same initial inhibition and subsequent increase of the growth rate is found (Fig. 20).
Thus, IAA can stimulate growth of coleoptiles even under anaerobic conditions if GTP or ITP is present at the same time.
4.
The results support the following hypothesis (Fig. 21): auxin acts cooperatively with GTP (ITP) as an effector of a membrane-bound, anisotropic ATPase or proton pump. This pump, activated by auxin, utilizes respiration energy (ATP or other nucleoside triphosphates) to raise the proton concentration in a compartment at the cell wall. This event leads to an increase in the activity of enzymes softening cell walls and thus triggers cell elongation. The transport or secretion of protons into the cell wall compartment should be compensated by a flow of cations into the interior of the cytoplasm or by a flow of anions to the cell periphery, thus causing secondary auxin effects.
The shape of an organism relies on a complex network of genetic regulations and on the homeostasis and distribution of growth factors. In parallel to the molecular control of growth, shape changes also involve major changes in structure, which by definition depend on the laws of mechanics. Thus, to understand morphogenesis, scientists have turned to interdisciplinary approaches associating biology and physics to investigate the contribution of mechanical forces in morphogenesis, sometimes re-examining theoretical concepts that were laid out by early physiologists. Major advances in the field have notably been possible thanks to the development of computer simulations and live quantitative imaging protocols in recent years. Here, we present the mechanical basis of shape changes in plants, focusing our discussion on undifferentiated tissues. How can growth be translated into a quantified geometrical output? What is the mechanical basis of cell and tissue growth? What is the contribution of mechanical forces in patterning?
Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact.
Plants are under tremendous mechanical forces generated by turgor pressure. How do these forces mediate growth and development? In order to answer this question, it is necessary to understand the mechanics of growth and morphogenesis. In this 'mathless' tutorial, the concepts of strain, mechanical stress and buckling are reviewed and illustrated with recent work on leaf shape, on leaf vasculature, and on organogenesis at the shoot apical meristem.
Many genes and molecules that drive tissue patterning during organogenesis and tissue regeneration have been discovered. Yet, we still lack a full understanding of how these chemical cues induce the formation of living tissues with their unique shapes and material properties. Here, we review work based on the convergence of physics, engineering and biology that suggests that mechanical forces generated by living cells are as crucial as genes and chemical signals for the control of embryological development, morphogenesis and tissue patterning.
Miniature high speed label-free cell analysis systems have yet to be developed, but have the potential to deliver fast, inexpensive and simple full blood cell analysis systems that could be used routinely in clinical practice. We demonstrate a microfluidic single cell impedance cytometer that performs a white blood cell differential count. The device consists of a microfluidic chip with micro-electrodes that measure the impedance of single cells at two frequencies. Human blood, treated with saponin/formic acid to lyse erythrocytes, flows through the device and a complete blood count is performed in a few minutes. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from CD antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Tests with patient samples showed 95% correlation against commercial (optical/Coulter) blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.
Mechanotransduction research has focused historically on how externally applied forces can affect cell signalling and function. A growing body of evidence suggests that contractile forces that are generated internally by the actomyosin cytoskeleton are also important in regulating cell behaviour, and suggest a broader role for mechanotransduction in biology. Although the molecular basis for these cellular forces in mechanotransduction is being pursued in cell culture, researchers are also beginning to appreciate their contribution to in vivo developmental processes. Here, we examine the role for mechanical forces and contractility in regulating cell and tissue structure and function during development.
Plant cells encase themselves within a complex polysaccharide wall, which constitutes the raw material that is used to manufacture textiles, paper, lumber, films, thickeners and other products. The plant cell wall is also the primary source of cellulose, the most abundant and useful biopolymer on the Earth. The cell wall not only strengthens the plant body, but also has key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. Recent discoveries have uncovered how plant cells synthesize wall polysaccharides, assemble them into a strong fibrous network and regulate wall expansion during cell growth.
Direct measurements of the volumetric elastic modulus, in, of cells of a higher plant were performed on the epidermal bladder cells of Mesembryanthemum crystallinum using a pressure probe technique. Measurements on giant algal cells (Valonia, Nitellopsis) are given for comparison. Giant celled algae and M. crystallinum bladders have elastic moduli, in, which depend strongly on turgor pressure, P, and on cell volume, V. The in values of Mesembryanthemum bladders range between 5 bar at zero pressure and 100 bar at full turgor pressure (3-4 bar). in increased with cell size (volume) at a given turgor pressure, and this volume dependence was pronounced more in the high pressure range. From the in (P) characteristics, complete volume-pressure curves were obtained for Mesembryanthemum bladders and giant algal cells. The results suggest that the in (P) and in (V) characteristics of all plant cells are similar. The significance of the pressure and volume effects for the water relations and growth processes of plant cells is discussed briefly.